This lesson is broken up into 5 parts and time is required
for the cultures to grow before you can isolate and stain them. Parts I and II
can be done in the same day within a 45 minute Lab period if students have been
given a pre- lab session on how to do these steps the day before.

Aim:How do we Culture Bacterial colonies and prepare
them for identification?

Sterile collection tubes
filled with 20 mL of sterile water (for a back-up in case you need to
re-isolate your bacteria samples)

A black permanent marker

Proper receptacle for
disposing of swabs, tubes, gloves, and plates after use

Procedure:

Determine beforehand where you'd like to sample for
bacterial cultures. Make sure you get permission, and discuss what kinds of
bacteria you might expect to find in each location.

At each location, put on a pair of gloves. Dip a
sterile swab into the dirt, water, or other substance to be sampled. Lightly
rub the end of the swab onto a sterile agar plate like you're painting a
line onto the surface of the agar. This will give you a mixed culture of
microbes.

Carefully dip the same swab into your water-filled
tube - this will give you a backup in case your first plate culture doesn't
work out.

Mark your plate with the date, location, and your
initials to keep track of your culture.

Part II: Isolating a Pure Bacterial Culture

Lab Materials:

Sterile swabs or toothpicks

Bunsen burner

Metal inoculating loop

Incubator (set at 37 degrees Celsius)

Extra agar plates for practicing your technique

Procedure:

From the line streaked onto the surface of your plate
(the mixed culture of microbes), you can try one of two techniques to
separate them:

Use a second sterile cotton swab or a sterile
toothpick to touch a portion of the line on your plate and gently streak
it across the plate in a zigzag pattern. Or:

Back at the lab, take a sterile inoculating loop
(loop is made sterile by passing it through flame from Bunsen burner
then cooled) and dip the loop into the culture of microbes. Then streak
this in a pattern over the surface of the agar plate. The inoculating
loop is sterilized following each streak series. As the pattern is
traced, bacteria are rubbed off the loop onto the medium. The last cells
to be rubbed off the loop should be far enough apart to grow into
isolated colonies.

2. Streaked plates are then
incubated overnight at 37 degrees Celsius. Selected colonies can then be picked
up from these plates with a sterile inoculating loop and transferred to separate
agar plates or culture tubes (such as typtic soy agar slants or tubes filled
with Luria broth) to form a pure culture. Once you have obtained a pure
bacterial culture, you are ready to stain your microorganism.

Part III: Staining Bacterial Colonies

Lab Materials:

Glass microscope slide

Inoculating loop

Bunsen burner

Distilled water

Crystal violet dye

Gram's iodine solution

Alcohol

Safranin

Procedure:

After pure culture is obtained, microorganism is fixed
to a microscope slide using an inoculating loop as follows: flame loop then
allow it to cool, dip loop into pure culture medium then smear it onto
microscope slide.

Allow smear to air dry. Smears can also be heat fixed
by carefully passing the slide through flame.

Cover the smear with crystal violet dye for 30
seconds. Because the purple stain imparts its color to all cells, it is
called a primary stain.

Rinse the purple dye off with distilled water then
cover the smear with Gram's iodine for 10 seconds.

Rinse gently with distilled water. Decolorize the
smear with alcohol.

Rinse with distilled water then cover the smear with
safranin for 30 seconds. Rinse the smear with distilled water and blot the
slide dry.

Observe slide under a microscope. Bacteria that
are gram negative will appear pink while those that are gram positive will
appear purplish.

Part IV: Identifying Bacterial Species

This kit is one
that I plan to try this year in order to help run some tests to identify what
the bacterial colonies might be.
Otherwise there are different resources for identifying and categorizing
bacteria based on size and color, but there are so many species this will become
difficult.